Although the regulation of gene expression has been intensively studied in the yeast S. cerevisiae, much about this process remains unknown. This is exemplified by our inability to predict, as opposed to explain, the expression pattern of any gene given its promoter sequence. Our long-term goal is to provide a comprehensive map of the S. cerevisiae gene regulatory network that can be used to develop predictive models of gene expression. The first task is to complete the catalog of transcription factors and their binding sites. We will use a combination of existing in vitro and in vivo methods to accomplish that goal. We will identify the binding sites of the more than 100 transcription factors of yeast whose specificity remains unknown (Aim 1) using electrophoretic gel mobility shift assays, a yeast one-hybrid assay, and a novel method to probe protein microarrays with DMA oligonucleotides. We will then develop comprehensive weight matrices of the binding sites of yeast transcription factors (Aim 2) using a novel implementation of the SELEX method we have developed. These results will be extended by determining the in vivo targets of selected transcription factors (Aim 3) using genome-wide chromatin immunoprecipitation (ChlP-Chip). We expect that the combination of these approaches will enable us to determine the binding sites and target genes of nearly all transcription factors of yeast. We will then attempt to learn the architectural principles of yeast promoters by determining how transcription factor binding sites contribute to gene expression. By creating large libraries of potential gene promoters in which a set of binding sites have been randomly distributed, we can ascertain the combinations of binding sites that determine specific expression patterns. This approach will be initially developed and tested using a few well characterized binding sites; we expect it will provide a general tool for more comprehensive studies of the logic of gene regulation. [unreadable] [unreadable] [unreadable]